新血管生成抑制素缺氧诱导表达特异抑制肿瘤生长和转移

In this study, we extracted mRNA from human fetal liver tissue, and amplified endostatin and angiostatin genes cDNA by means of RT-PCR. The amplified DNA sequences was confirmed to be identical to the published. Prokaryotic Endostatin expression vectors were constructed. Human endostatin protein was expressed in E.Coli and purified. In vitro study showed endostatin protein suppressed the growth of HUVEC human endothelial cells. Endostatin gene ligated to interleukin-3 signal sequence, and cloned into adenoviral vector pAdCMV and then generated recombinant adenovirus AdIL3Endo. AdIL3Endo was employed to infect human colorectal carcinoma SW620. Endostatin protein was thereafter detected in the supernatants of the infected cells by means of RT-PCR and Western Blot. This protein was also demonstrated exhibiting strong growth inhibition to HUVEC cells. This recombinant adenovirus showed significant growth inhibition effect to SW620 model in nude mice. CD31 immunostaining results showed that mechanism of the tumor inhibition was due to anti-angiogeneic effect of endostatin. In order to enhance the effect of anti-angiogeneic effect of endostatin, we constructed a recombinant adenovirus encoding endostatin-soluble VEGI. This adenovirus showed more strong growth inhibition effect to human gastric cancer SGC7903 model in nude mice than AdIL3Endo did. During this study, we confirmed that the anti-angiogenesis effect of Angiostatin is not as good as expected. The anti-angiogenesis result of Interferon-β was very apparent and even stronger than endostatin, as measured by CD31 immunostaining. We found recombinant adenoviruses encoding Endostatin, class I interferons as well as soluble VEGF receptor exhibited strong antiangiogeneic effect following transferred into the tumor tissues, and also significantly delayed the death of the tumor bearing animals caused by tumor metastasis. We found that the induction of inducible nitric oxide synthetase (iNOS) was involved in the major mechanism of antiangiogenesic effect of Interferon-β, resulting in the increase of nitric oxide (NO). NO served as second messenger inhibiting hypoxia-inducible mechanism of angiogenesis, and inducing the apoptosis of new endothelial cells of neovascularization in the tumor bodies. As a matter of fact, none of the anti-angiogenesis molecules was found to be able to completely blocking tumor neovascularization and stopping the enlargement of the metastatic locus. Most of the tumor bearing animals finally died of cancer metastasis. Tumor induced angiogenesis is a complex patho-physiological process, key point of whom is currently not elucidated. Endostatin, IFNb and sFlt-1 inhibit tumor neovascurization in vivo by different mechanisms, which might be treated as the foundation of collaborative effect to anti-angiogenesis. In this study, we confirmed that the anti-angiogenesis effect of combining Endostatin and IFNb was much better than each of them used individually. Conclusions drown from this study served as theoretical basis of the mechanism study of anti-neovascurization and antitumor metastasis based on inhibition of angiogenesis.

建立安全有效的抗实体瘤血管生成基因治疗。在腺病载体中用VEGF e/p分别调控HIF-VP16重组基因和Endostatin、Endostatin-sFLT1基因。用缺氧诱导机制正反馈激活VEGFe/p使3种血管生成抑制因子在肿瘤组织中高效特异表达。在体外和裸鼠模型中系统研究基因诱导表达和抗人肿瘤新血管生成、肿瘤生长和转移效果。为抗新血管生成基因治疗控制肿瘤生长和转移奠定理论基础。